PROJECT SUMMARY: Ion channel regulation by variable auxiliary subunits is a major mechanism in generating diversity of ion channel function and thus variability in electrical signaling in different tissues and cells. Large conductance, calcium- and voltage-activated potassium (BK) channels are ubiquitously expressed and critically involved in various cellular and physiological processes including regulation of neuronal excitability and synaptic transmission and control of contractile tone of almost all types of smooth muscle cells. BK channels are diversified in structure and function by the presence of several tissue specific auxiliary ? and ? subunits. Since our initial identification of the leucine-rich repeat containing (LRRC) membrane protein LRRC26 as the BK channel auxiliary ?1 subunit, an increasing number of LRRC proteins, among hundreds of LRRC proteins in the human protein database, have been found to function as regulatory proteins of ion channels. Currently, little is known about the mechanisms underlying ion channel regulation by most regulatory LRRC proteins. The BK channel auxiliary ?1 subunit have characteristics of an atypical ?all-or-none? modulatory action, an exceptionally large capability in affecting the BK channel?s voltage-gating, a predicted major effect on the allosteric coupling between the voltage sensor activation and channel pore-opening, and a competitive relationship with a small molecule BK channel activator mallotoxin (rottlerin). Based on our previous and current studies, we hypothesize that the auxiliary ?1 subunit modulates BK channels via a central intramembrane mechanism and also subsidiary extracellular and intracellular mechanisms. To test our hypothesis and elucidate the molecular mechanisms of BK channel modulation by auxiliary ? subunits, we propose to pursue the following 3 specific aims: 1) determine the molecular basis underlying an atypical ?all-or- none? action of the ?1 subunit on BK channel modulation; 2) determine the molecular mechanisms of BK channel modulation by the ?1 subunit involving transmembrane domains; 3) determine the molecular mechanisms of BK channel modulation by the ?1 subunit and mallotoxin involving cytoplasmic domains. The proposed research in this grant application is designed to systematically investigate the biochemical and biophysical mechanisms governing BK channel regulation by the auxiliary ?1 subunit and mallotoxin. The knowledge obtained in this study could be applicable to ion channel regulation by other regulatory LRRC proteins. The findings from the proposed studies will shed light on mechanisms of BK channel voltage gating and provide in-depth understanding of the ?1 subunit?s atypical ?all-or-none? action and exceptional capability in modulating BK channel voltage-gating. They will also help in creation of novel therapeutic reagents targeting BK channels in treatment or prevention of neurobiological, cardiovascular, and other types of disorders and diseases.